The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In the Sanger's sequencing method DNA fragments are synthesised by DNA polymerase which incorporates deoxynucleotide monomers into a polymeric complementary copy of a template DNA strand. An oligonucleotide primer is used to initiate the synthesis of the new DNA stand from the template DNA at a single specific location (Sanger, F., Nicken, S., Coulson, A. R., 1977, PNAS, 74, 5463). Four separate reactions are performed, each containing a carefully controlled ratio of one particular 2′,3′-dideoxynucleoside 5′-triphosphate (ddNTP) to the corresponding 2′-deoxynucleoside 5′-triphosphate (dNTP), and the other dNTPs. Once incorporated into a growing DNA strand, the dideoxynucleotide is not able to form a phosphodiester bond with the next incoming dNTP, and the growth of that particular DNA chain stops. Thus, a series of strands is obtained, the lengths of which depend on the location of ddNTP.
In order to detect these oligomers, each fragment must be labelled in some manner. Traditionally, labelling has been accomplished with radioisotopes, such as 32p or 35S prior of during the polymerase reaction, i.e. using either a radioisotopically labelled primer of dNTPs. Although the radioactive detection is very sensitive, it has intrinsic hazard, expense and problems associated with the short half-lives of the radioactive isotopes commonly used.
Three different methods can be used to avoid radioactive detection:
(i) the use of a primer labelled with a detectable group, such as a chemiluminescent dye (Hunkapiller, T., Kaiser, R. J., Koop, B. F., Hood, K. L., 1991, Science, 254, 59, Smith, L. M., Fung, S, Hunkapiller, M. W., Hunkapiller, T. J., Hood, L. E., 1985, Nucleic Acids Res., 13, 2399, Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S. B. H., Hood, L. E., 1986, Nature, 321, 674);
(ii) the use of ddNTPs tethered to detectable groups as terminators of DNA synthesis (Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., Baumeister, K. 1987, Science, 238, 336, Lee, L. G., Connell, C. R., Woo, S. L., Cheng, R. D., McArdle, B. F., Fuller, C. W., Hallolan, N. D., Wilson, R. K., 1992, Nucleic Acids Res., 20, 2471);
(iii) the use mass spectrometry to analyse DNA fragments (Fizgerald, M. C., Zhu, L., Smith, L. M., 1993, Rapid Commun. Mass Spectrom., 7, 895).
The labeled dNTPs ought to be almost as good substrates to DNA polymerases as normal dNTPs. Since DNA polymerases are very sensitive to structural changes of their substrates, the selection of methods to attach non-radioactive markers into dNTPs are rather limited. It has already shown that the well-known terminators of DNA synthesis, 2′,3′-dideoxy-3′-amino NTPs (Chidzeavadze, Z. G., Beabealashvilli, R. Sh., Anttrazhev, A. M., Kukhanova, M. K., Azhayev, A., Krayevsky, A. A., 1984, Nucleic Acids Res., 12, 1671) are not substrates of DNA polymerases when bulky reporter groups are attached to their 3′-amino function (Herrlein, M. K., Konrad, R. E., Engels, J. W., Holletz, T., Cech, D., 1994, Helv. Chim. Acta, 77, 586). Analogously, while 2′-amino-2′-deoxy-ara adenosine TP is an effective terminating substrate, introduction of bulky substituent to the 2′-position causes total loss of substrate properties of the molecule (Alexandriva, L. A., Sharkin, Yu. A., 1993, Collect. Czech. Chem. Commun. Spec. issue, 58, 113). These problems can be solved by attaching the label to 3′-position of the carbohydrate moiety in the end of a flexible tether arm (Hovinen, J., Azhayeva, E., Azhayev, A., Guzaev, A., Lönnberg, H., 1994, J. Chem. Soc. Perkin Trans 1, 211). In such manner the bulky reported group may be kept distant from the catalytic centre of the polymerase enzyme, while the base residues remain unmodified. Furthermore, the flexible arm should not severely restrict the conformational motion of the sugar ring upon binding to enzyme.
A method reported by Prober et al. (Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J., Cocuzza, A. J., Jensen, M. A., Baumeister, K. 1987, Science, 238, 336) consists of the preparation of ddNTPs bearing four different labels attached to their base moieties. Since their fluorescence spectra differ from each other all four terminating triphosphates can be used in a single sequencing reaction. The label is attached covalently to the nucleobase via a rigid propargylamine linker at C5 of the pyrimidine nucleosides and at C7 of 7-deazapurine nucleosides, e.g. at positions which are not involved in the formation of normal Watson-Crick base pairs. However, these label molecules used are organic dyes which suffer from commonly known drawbacks such as Raman scattering, concentration quenching and low water solubility.
A solid-phase method, called minisequencing, has been introduced for detection of point mutation of DNA (Syvänen, A.-C., Allto-Setälä, K., Harju, L., Söderlund, H., 1990, Genomics, 8, 684, Jalanko, A., Kere, J., Savilahti, E., Schwartz, M., Syvänen, A.-C., Söderlund, H., 1992, Clin. Chem, 38, 39) The method involves hybridization of immobilized single-stranded DNA with a primer that ends immediately before the site of mutation, and elongation of the chain with a single labeled deoxyriobonucleoside 5′-triphosphate. Parallel runs with each of the four possible nucleotides enable identification of the mutated base. Thus far, practically only radioactive detection methods have been used. Application of fluorescence techniques has been suffered from drawbacks such as low detection sensitivity and problems assosiated with the properties of the labeled triphosphates to act as good substrates for DNA polymerases.
The unique properties of lanthanide(III) chelates such as strong long-decay time luminescence make them ideal markers for numerous applications. Furthermore, large Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Sm, Dy) in the analysis. Time resolved fluorimetric assays based on lanthanide chelates have found increasing applications in diagnostics, research and high throughput screening. The heterogenous DELFIA technique (EP 0139675 B1; U.S. Pat. No. 4,808,541; EP 0298939 B1; U.S. Pat. No. 6,127,529; U.S. Pat. No. 4,565,790; WO 03/076939; Hemmilä I., Dakubu, S., Mukkala, V.-M., Siitari, H., Lövgren, T., 1984, Anal. Biochem. 137, 335; FI Pat. Appl. 20065030) is applied in assays requiring exceptional sensitivity, robustness and multi-label approach. The development highly stable and luminescent lanthanide(III) chelates (Hemmilä, I.; Mukkala, V.-M. 2001, Crit. Rev. Clin. Lab. Sci. 38, 441) has enabled the use of homogenous assay technologies based on time resolution. The different photochemical properties of europium, terbium, dysprosium and samarium chelates enable development even multiparametric homogenous assays. Accordingly, a number of attempts have been made to develop new highly luminescent chelate labels suitable for time-resolved fluorometric applications. These include e.g. stabile chelates composed of derivatives of pyridines [U.S. Pat. No. 4,920,195; U.S. Pat. No. 4,801,722; U.S Pat. No. 4,761,481; PCT/FI91/00373; U.S. Pat. No. 4,459,186; EP A-0770610; Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [U.S. Pat. No. 5,216,134], terpyridines [U.S. Pat. No. 4,859,777; U.S. Pat. No. 5,202,423; U.S. Pat. No. 5,324,825] or various phenolic compounds [U.S. Pat. No. 4,670,572; U.S. Pat. No. 4,794,191; Ital Pat. 42508 A789] as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [U.S. Pat. No. 5,032,677; U.S. Pat. No. 5,055,578; U.S. Pat. No. 4,772,563] macrocyclic cryptates [U.S. Pat. No. 4,927,923; WO 93/5049; EP-A-493745] and macrocyclic Schiff bases [EP-A-369000] have been disclosed. Recently, development of neutral, highly luminescent stable europium, terbium, samarium and dysprosium chelates based on azamacrocycles has been disclosed [U.S. patent application Ser. No. 10/928,143; U.S. patent application Ser. No. 11/004,061].
However, the use of lanthanide(III) chelates as reporter groups in terminating substrates for DNA polymerases has not been suggested in prior art.